1. Field of the Invention
The present invention relates to the binding of antigens to a solid phase material for use in a diagnostic assay. In particular, the invention increases the effective amount of antigen that can be bound to the solid phase, for example, to a thin microporous sheet of nitrocellulose, thereby making more antigenic sites available for antibody binding in an immunoassay.
2. Background of the Invention
U.S. Pat. No. 3,720,760 discloses that certain immunogenic substances, called allergens, can give rise to allergic reactions in the form of asthma, hay fever, and the like, and that the blood of a patient in whom a given allergen causes an allergic reaction usually contains low concentrations of immunoglobulins, called reagin-immunoglobulins (now usually called "IgE", which term is subsequently used herein), which are directed specifically against that allergen. The patent discloses a test for sensitivity to allergens which involves injecting given allergens into the skin of a patient; a skilled observer then assesses the degree of sensitivity to each of the allergens on the basis of the observed reaction (reddening or swelling of the skin) caused by each allergen. The patent also discloses an "in vivo" test, where the patient inhales an allergen in the form of an aerosol, and the patient is deemed to be sensitive to any allergen that causes hay fever, asthma or like symptoms.
The patent further discloses an in vitro method for determining the presence of IgE in a body fluid. The method involves binding an allergen to fine particles of a copolymer, e.g., a dextran-epichlorohydrin copolymer, by treating the particles with cyanogen bromide, and suspending the particles and an allergen in an aqueous medium. A body fluid to be tested for the presence of IgE directed against that allergen is then contacted with the allergen bound to the copolymer. The product of step (2) is then brought into contact with radio-labeled antibodies which will bind to IgE, if any, that has become bound to the allergen that is bound to the copolymer. The radiation emitted from the solids of step (3), the liquid of step (3), or both can then be measured.
The covalent coupling of antigens, including allergens, to a solid phase was used to prevent or inhibit their removal from the solid phase during the assay procedure. U.S. Pat. No. 4,597,999 describes the covalent coupling of two molecular species to one another, using cross-linking agents having at least two functional groups which are subject to independent activation. Examples of such cross-linking agents include 4-methylazidobenzidimate and N-hydroxysuccinimidyl-azidobenzoate. These cross-linking agents couple spontaneously in the dark to available amino groups, as in aminopropyl glass, aminophenyl glass and aminohexylagarose, and when activated by irradiation with light of a suitable wavelength, these agents also couple with a ligand such as a drug, digoxin, a steroid, or a protein.
U.S. Pat. No. 4,425,434 describes the use of biologically active substances to fill the pores of porous titania spheroids, porous calcium phosphate spheroids, porous zirconia spheroids or similar porous support material, and that the biologically active substance can then be immobilized in the pores by precipitation and cross-linking. The biologically active substance can be a proteinaceous substance, such as an enzyme.
It has been found, however, that the covalent coupling procedures are costly to perform and time consuming. In addition, some coupling procedures can decrease the sensitivity of the assay.